Laboratory of Functional Genomics and Proteomics of Model System Rodolfo Negri Sapienza Università di Roma The Transcriptional Response to Irradiation: Time to Move the Focus from Cellular to Tissutal Level Melodi Workshop 2013
Radiat Res. 2001 Oct;156(4):379-87. Cell cycle arrest determines the intensity of the global transcriptional response of Saccharomyces cerevisiae to ionizing radiation. De Sanctis V, Bertozzi C, Costanzo G, Di Mauro E, Negri R.
Many transcriptional modulations are not enclosed in a radiation damage signature: Cell Cycle regulated genes responding to cell cycle variations Stress responsive genes
General features of the transcriptional response of mammalian cells to irradiation (work of several different groups) The transcriptional response to irradiation is Strongly dose-dependent The transcriptional response to irradiation is Strongly cell type dependent
A system Biology approach to the Transcriptional Response to radiation
Journals databases
34 Genes modulated at least 1.5 fold in at least 30% of the 208 conditions OH et al. 2012 Rashi-Elkeles et al. 2011
Cell cycle process (Fold enrichment = 9.6; p = 5.3 E-4) Mitotic cell cycle (Fold enrichment = 11.7; p = 8.2 E-4) Negative regulation of apoptosis (Fold enrichment = 7.6; p = 1.3 E-2)
Expression of some general responding genes is predictive of Radiosensitivity D 0 = 37% survival reduction at dose < 90 cgy
E2F4 controls the generally down-regulated gene cluster Amundson et al.2008 Cancer Res. 68, 415-424
Searching for conserved motifs in the generally up-regulated gene cluster
Motif 1
Motif 1 Name Alt. Name MA0024.1 E2F1 Database p-value JASPAR_CORE_2009_ve rtebrates.meme 3.28253e-05 E-value 0.017463 q-value 0.0347387 Motif 1 is hyghly similar to the E2F1 consensus sequence
Plesca et al. 2007 E2F4 Function in G2 Maintaining G2-Arrest to Prevent Mitotic Entry with Damaged DNA Ionizing Radiation ATM (Chk2) A mechanism of regulation partially independent from P53, ATM and ATR transduction pathways? P130 E2F4 Rb E2F1 Induction of apoptosis Regulators and other target genes: MEF2A; CDKN1A; TNFAIP3; ATF3; LAMC1; FMR2 Nuclear Translocation and transcriptional repression of mitotic target genes: BUB 1,3; PLK1; CENP-A; CENP-E; AURKA; AURKB; CKS2; KIF23; others. Regulation of Apoptosis and DNA Repair Maintenance of G2 arrest
The transcriptional response of mammalian cancer cells to irradiation is dominated by a cell cycle signature which is strongly attenuated in non cancer cells and tissues. Bufalieri et al., 2012 Giusti et al., 2013 in press
Cdkn1A coding for P21 is the most generally responsive gene
General responsive genes are differently modulated in ploriferative versus non ploriferative tissues Bufalieri et al., 2012 Giusti et al., 2013 in press
Reconstructing Pathways of Response to Radiation at Tissue Level At cellular level: At tissue level: Sensor? (cells; extracellular matrix) Signalling? (hormones; cytokines; mirnas; ROS) Effectors? (Stem cells; immunity system) Tissue Response and Repair/Necrosis Systemic Signalling and Response
The tissutal transcriptional response to irradiation is different from the cellular response and strongly dose-dependent Fratini et al., 2011
A schematic leptin pathway. Leptin is released mainly from white adipose tissues and from peripheral organs as shown. Leptin exerts its principal effects in hypothalamus promoting loss body weight, satiety and energy expenditure. Peripheral effects: -Mitogenic effect on skin -Stimulation of T cells and monocytes to release citokines (GM-CSF; TNF-a; IL6) -Pro-angiogenic effects on endothelial cells
Cestelli Guidi et al. 2012 Increase of Leptin mrna observed at the indicated doses and times from Neutron irradiation by DNA microarrays (A) or RT-qPCR (B).
Leptin increase is observed in adipocytes and epidermis at 24h from irradiation
Cestelli Guidi et al. 2012 Reduction of lipid content in irradiated skin See Cestelli Guidi et al. 2012 for details
? 0.2 or 1 Gy Neutron irradiation An hypotetical mirna regulatory network for leptin LEPRC mir706 LEP mir29a mir294 mir29b mir29c mir30e mir671 mir30c mir19a LEPRA LEPRB mir206 mir19b Keratinocytes Proliferation BLOOD Target Rank <30 in Target Scan and MirDB
Log2 Irradiated / Control Fig. 3B Correlation between Mir modulation observed in microarray profiling and RT-PCR validation experiments 2.00 1.00 0.00-1.00-2.00-3.00 Log2 PCR Log2 MA -4.00-5.00-6.00-7.00 mir-294 mir-294 mir-206 mir-206 mir-29b mir-29b mir-30c mir-30c 0.2Gy 6h 1 Gy 6h 0.2Gy 6h 1 Gy 6h 0.2Gy 6h 1 Gy 6h 0.2Gy 6h 1 Gy 6h R = 0.968 P<7.6 x 10-5
Searching for transcriptional regulators of Leptin in Response to ionizing radiation
Human Lep Does AP1 TF control the induction of Leptin mrna in response to irradiation?
Conclusions and perpectives: - Leptin is transcriptionally induced in skin during wound healing and after Irradiation - In both conditions Leptin accumulates in adipocytes and epidermis - A peak is observed in the blood stream during wound healing (to be confirmed after irradiation) - A network of radiation-modulated mirnas could regulate Leptin and Leptin receptors Open questions: - What is the mechanism of Leptin regulation upon irradiation? - What is the meaning of this regulation: - Is it limited to a mitogenic effect on keratynocytes through the Jak/Stat pathway? - Or is it able to trigger Systemic Effects? -Is the activation of Leptin circuit a prodromal response to irradiation? -Is the activation of Leptin circuit dose and/or radiation quality-dependent? -Is the activation of Leptin circuit dependent on crosstalk between tissutal components? -Which signals are involved? CAN LEPTIN REPRESENT A GENERAL ALARM TRANSMITTED BY THE SENSOR SKIN TO THE CENTRAL NERVOUS SYSTEM AND TO THE IMMUNE SYSTEM?
V. Licursi F.Bufalieri F. Chiani V. Lanza C. Iannone M. Etna N. Giusti S. Vincenti C. Presutti Laboratory of Functional Genomics and Proteomics of Model Systems Sapienza University of Rome C.Mannironi IBPM CNR R. Amendola, ENEA, BAS BIOTEC MED, Roma, Italy; E. Fratini T. Castrignanò, Caspur, Roma, Italy P. D Onorio De Meo, M. D Antonio, A. Paoletti This work has been partially supported by Agenzia Spaziale Italiana A.Marcelli M. Cestelli Guidi INFN, LNF, Frascati, Italy